DNA molecules encoding human tissue plasminogen activator variants with decreased clearance, vectors, and host cells

ABSTRACT

Biologically active tissue plasminogen activator (t-PA) variants with decreased clearance as compared to wild-type t-PA are prepared, including variants that have one or more amino acid alterations in at least the kringle-1 and/or kringle-2 domain(s) of the molecule. DNA sequences can be prepared that encode the variants, as well as expression vectors incorporating the DNA sequences and host cells transformed with the expression vectors. The variants may be used in pharmaceutical preparations to treat a vascular disease or condition, or to prevent fibrin deposition or adhesion formation or reformation in mammals.

This is a continuation of co-pending application Ser. No. 07/560,515filed on Jul. 31, 1990 now abandoned.

BACKGROUND OF THE INVENTION

I. Field of the Invention

This invention is directed to particular tissue plasminogen activator(t-PA) variants, to methods for preparing these variants, and to methodsand compositions utilizing the variants in pharmaceutical compositions.Specifically, this invention relates to t-PA variants with modifiedamino acid sequences, including substitutions, within at least thekringle-1 or kringle-2 domains of t-PA that result in the variantshaving a decreased rate of clearance as compared to wild-type t-PA.

II. Description of Background and Related Art

Plasminogen activators are enzymes that cleave the peptide bond ofplasminogen between amino acid residues 561 and 562, converting it toplasmin. Plasmin is an active serine proteinase that degrades variousproteins including fibrin. Several plasminogen activators have beenidentified including streptokinase (a bacterial protein), urokinase(synthesized in the kidney and elsewhere and originally extracted fromurine), and human tissue plasminogen activator, termed t-PA (produced bythe cells lining blood vessel walls).

The mode of action of each of these plasminogen activators is somewhatdifferent. Streptokinase forms a complex with plasminogen or plasmingenerating plasminogen-activating activity, urokinase cleavesplasminogen directly, and t-PA interacts with both plasminogen andfibrin for optimal activity.

Due in part to its high fibrin specificity and potent ability todissolve blood clots in vivo, t-PA has been identified as an importantnew biological pharmaceutical for treating vascular diseases such asmyocardial infarction.

A substantially pure form of t-PA was first produced from a naturalsource and tested for in vivo activity by Collen et al., U.S. Pat. No.4,752,603 issued Jun. 21, 1988 (see also Rijken et al., J. Biol. Chem.,256:7035 [1981]). Pennica et al. (Nature, 301:214 [1983]) determined theDNA sequence of t-PA and deduced the amino acid sequence from this DNAsequence (see U.S. Pat. No. 4,766,075 issued Aug. 23, 1988).

Human native t-PA has potential N-linked glycosylation sites at aminoacid positions 117, 184, 218, and 448. A high mannose oligosaccharide ispresent at position 117 and a complex oligosaccharide is present atpositions 184 and 448. Sites 117 and 448 appear to always beglycosylated, while site 184 is thought to be glycosylated in aboutfifty percent of the molecules. The partial glycosylation pattern atposition 184 may be due to site 184 being situated in an unexposedregion of the molecule. The t-PA molecules that are glycosylated atposition 184 are termed Type I t-PA, and the molecules that are notglycosylated at position 184 are termed Type II t-PA. Position 218 hasnot been found to be glycosylated in native t-PA.

Research on the structure of t-PA has identified the molecule as havingfive domains. Each domain has been defined with reference to homologousstructural or functional regions in other proteins such as trypsin,chymotrypsin, plasminogen, prothrombin, fibronectin, and epidermalgrowth factor (EGF). These domains have been designated, starting at theN-terminus of the amino acid sequence of t-PA, as the finger (F) domainfrom amino acids 1 to about 44, the growth factor (G) domain from aboutamino acids 45 to 91 (based on homology with EGF), the kringle-1 (Kl)domain from about amino acids 92-173, the kringle-2 (K2) domain fromabout amino acids 180 to 261, and the serine protease (P) domain fromabout amino acid 264 to the carboxyl terminus at amino acid 527. Thesedomains are situated essentially adjacent to each other, and some areconnected by short "linker" regions. These linker regions bring thetotal number of amino acids of the mature polypeptide to 527, althoughthree additional residues (Gly-Ala-Arg) may be found at the aminoterminus and are probably the result of incomplete precursor processingof the molecule.

Each domain is believed to confer certain biologically significantproperties on the t-PA molecule. The finger domain is thought to beimportant in the high binding affinity of t-PA to fibrin. This activityappears to be important for the high specificity that t-PA displays withrespect to clot lysis at the locus of a fibrin-rich thrombus. Thekringle-1 and kringle-2 domains also appear to be associated with fibrinbinding and with the ability of fibrin to stimulate the activity oft-PA. The serine protease domain is responsible for the enzymaticactivity of t-PA which results in the conversion of plasminogen toplasmin. The t-PA molecule is often cleaved between position 275 andposition 276 (located in this serine protease domain) to generate the2-chain form of the molecule.

Natural t-PA has a plasma half-life of about six minutes or less whenadministered to patients in therapeutically effective amounts. Incertain situations, a six-minute half-life is desirable, as for example,in aggressive therapy of a life-threatening disease such as myocardialinfarction or pulmonary embolism. In these high-risk situations,patients who have significant or unrecognized potential for uncontrolledbleeding may be treated with t-PA. If such bleeding occurs, drugadministration can be stopped and the causative t-PA levels will rapidlydrop. Thus, treatment of these patients with a relatively short-livedform of t-PA is preferred.

Despite the profound advantages associated with natural t-PA as aclot-dissolving agent, it is not believed that the naturally occurringform of the protein necessarily represents the optimal t-PA agent underall circumstances. In some instances, such as treatment of deep veinthrombosis, treatment following reperfusion of an infarct victim,treatment of pulmonary embolism, or treatment using bolus injection, at-PA molecule with a longer half-life and/or decreased clearance isdesirable. Several variants of the wild-type t-PA molecule have beengenerated in attempts to increase half-life or decrease the clearancerate.

One method used to generate such t-PA variants has been to deleteindividual amino acids, partial domains, or complete domains from themolecule. For example, removal of part or all of the finger domain oft-PA as described in U.S. Patent Number 4,935,237 (issued Jun. 19, 1990)results in a molecule with decreased clearance, although it hassubstantially diminished fibrin-binding characteristics. Browne et al.(J. Biol. Chem., 263:1599 [1988]) deleted the region between amino acids57 and 81 and found the resulting variant to have a slower clearancefrom plasma. Collen et al. (Blood, 71:216 [1988]) deleted amino acids6-86 (part of the finger and growth domains) and found that this mutanthad a half-life in rabbits of 15 minutes as compared with 5 minutes forwild-type t-PA. Similarly, Kaylan et al. (J. Biol. Chem., 263:3971[1988]) deleted amino acids 1-89 and found that the half-life of thismutant in mice was about fifteen minutes as compared to about twominutes for wild-type t-PA. Cambier et al. (J. Cardiovasc. Pharmacol.,11:468 [1988]) constructed a variant with the finger and growth factordomains deleted and the three asparagine glycosylation sites abolished.This variant was shown to have a longer half-life than wild-type t-PAwhen tested in dogs. Variants with only the growth factor domain or thefinger domain deleted have also been demonstrated to have decreasedclearance rates in rabbits, guinea pigs and rats (Higgins and Bennett,Ann. Rev. Pharmacol. Toxicol., 30:91 [1990] and references therein).

Various deletions in the growth factor region have also been reported inthe patent literature. See EPO Publication Number 241,208 (deletion ofamino acids 51-87, and deletion of amino acids 51-173). See also EPOPublication Number 240,334 which describes the modification of mature,native t-PA in the region of amino acids 67-69 by deletion orsubstitution of one or more amino acids.

Another means to improve the clearance rate and/or half-life of t-PA hasbeen to complex the t-PA molecule with a second molecule. For example, at-PA-polyethylene-glycol conjugate has been reported to enhance the rateof clearance of t-PA, as reported in EPO 304,311 (published Feb. 22,1989). A monoclonal antibody to t-PA has been reported to increase thefunctional half-life of t-PA in vivo without decreasing its activity(see EPO 339,505 published Nov. 2, 1989).

A variety of amino acid substitution t-PA variants have been evaluatedfor their ability to decrease the clearance rate or increase thehalf-life of t-PA. The variant R275E (where arginine at position 275 ofnative, mature t-PA was substituted with glutamic acid) has been shownto have a clearance rate of about two times slower than that ofwild-type t-PA when tested in primates and rabbits (Hotchkiss et al.,Thromb. Haemost., 58:491 [1987]). Substitutions in the region of aminoacids 63-72 of mature, native t-PA, and especially at positions 67 and68, have been reported to increase the plasma half-life of t-PA (see WO89/12681, published Dec. 28, 1989).

Production of other substitution variants has focused on converting theglycosylation sites of t-PA to non-glycosylated sites. Hotchkiss et al.(Thromb. Haemost., 60:255 [1988]) selectively removed oligosaccharideresidues from the t-PA molecule, and demonstrated that the removal ofthese residues decreased the rate of clearance of t-PA when tested inrabbits. Removal of the high mannose oligosaccharide at position 117using the enzyme endo-β-N-acetylglucosaminidase H (Endo-H) resulted in arate of clearance that was decreased about two fold. Oxidation of nearlyall oligosaccharide residues using sodium periodate resulted in a rateof clearance nearly three fold lower than wild-type t-PA. Theseresearchers also generated the t-PA variant N117Q (wherein asparagine atposition 117 of native, mature t-PA was substituted with glutamine) toprevent glycosylation at position 117. The clearance rate of thisvariant was lower than wild-type t-PA. See also EP 238,304 publishedSep. 23, 1987 and EP 227,462 published Jul. 1, 1987.

An additional approach to produce t-PA variants with extendedcirculatory half-life and slower clearance has been to add glycosylationsites to the molecule. As examples of this approach, positions 60, 64,65, 66, 67, 78, 79, 80, 81, 82, and 103 have been substituted withappropriate amino acids to create molecules with glycosylation sites ator near some of these residues (see WO 89/11531, published Nov. 30, 1989and U.S. Ser. No. 07/480691, filed Feb. 15, 1990 now abandoned).

While some of the above cited work has resulted in generation of t-PAvariants with increased half-life or decreased clearance rates, in manyinstances the activity, solubility, and/or fibrin-binding specificity ofthe molecule has been compromised. Thus, the known t-PA variants havenot possessed optimal characteristics.

Accordingly, it is an object of this invention to prepare t-PA variantswith decreased clearance rates that substantially retain biologicalactivity, solubility and/or fibrin specificity. Production of t-PAvariants with decreased clearance that possess any one or a combinationof these characteristics will improve the therapeutic value and efficacyof t-PA. A further object of this invention is to produce t-PA variantswith improved efficacy or pharmaceutical utility.

SUMMARY OF THE INVENTION

In accordance with the objects of this invention, t-PA variants areprovided that exhibit biological activity and have decreased clearancerates as compared to wild-type t-PA.

More specifically, the invention provides a t-PA amino acid sequencevariant with an alteration at position 94 or 95, or at positions 236,238, and 240, that exhibits biological activity and has a decreasedclearance as compared to wild-type t-PA.

In another preferred embodiment, the alteration at positions 94 or 95,or at positions 236, 238 and 240 is a substitution, and the substitutedamino acids are replaced with preferably alanine, glycine, serine orthreonine. In a most preferred embodiment, they are replaced withalanine, or in certain positions, glycine.

In other embodiments, the t-PA variants are altered at more than oneposition such as at positions 94 and 95, or at positions 94, 236, 238and 240, or at positions 95, 236, 238, and 240, or at positions 94, 95,236, 238, and 240. Preferably, the alteration will be an amino acidsubstitution, preferably with alanine, glycine, serine, or threonine,and most preferably with alanine, or at certain positions, glycine.

In another embodiment, the above described t-PA variants, substituted atpositions 94 or 95 or both, or at positions 236, 238, and 240, or atpositions 94, 236, 238 and 240, or at positions 95, 236, 238, and 240,or at positions 94, 95, 236, 238, and 240 are additionally altered atposition 103 and/or position 117 and the alteration is preferably anamino acid substitution with asparagine at position 103 and alanine orserine, or preferably glutamine, at position 117.

In other embodiments, the invention relates to a DNA sequence encodingthe variants described above, replicable expression vectors capable ofexpressing this DNA sequence in a transformed host cell, and transformedhost cells.

In yet another embodiment, the invention relates to a composition fortreating a vascular condition or disease comprising a therapeuticallyeffective amount of the t-PA variant in admixture with apharmaceutically acceptable carrier.

In still another embodiment, the invention provides a method of treatinga vascular disease or condition in a mammal comprising administering aneffective amount of the t-PA variant to the mammal.

In still another embodiment, the invention provides a composition forpreventing fibrin deposition or adhesion formation or reformationcomprising a therapeutically effective amount of the t-PA variant inadmixture with a pharmaceutically acceptable carrier.

In one other embodiment, the invention is directed to a method fortreating a mammal to prevent fibrin deposition or adhesion formation orreformation comprising administering to a site on the mammal ofpotential fibrin or adhesion formation an effective amount of the t-PAvariant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the amino acid sequence of the mature form of human t-PA.The amino acids are numbered starting from the amino terminus. The fivedomains, disulfide bridging, and the activation site where the moleculeis clipped into the two-chain form are indicated.

FIG. 2 diagrams the construction of pRK.t-PA. The human t-PA PA cDNA wasdigested with HindIII and BalI and inserted into the eukaryoticexpression vector pRK7 between the HinddIII and SmaI sites.

FIG. 3 is a plot of the amount of radiolabeled t-PA variant remaining inthe bloodstream of mice (measured in thousands of counts per minute perml of blood) versus time (in minutes). Radiolabelled t-PA variants weretypically 1000 cpm per nanogram of protein. Each variant assayed isindicated, as is wild-type t-PA.

DETAILED DESCRIPTION OF THE INVENTION I. DEFINITIONS

The terms "t-PA", "human t-PA", and "human tissue plasminogen activator"refer to human extrinsic (tissue-type) plasminogen activator having twofunctional regions consisting of a protease domain that is capable ofconverting plasminogen to plasmin and an N-terminal region believed tobe responsible for fibrin binding. These terms thus include polypeptidescontaining these functional domains as part of the amino acid sequenceof the polypeptide. Biologically active forms of t-PA may be produced byrecombinant cell culture systems in forms comprising the two functionalregions of the molecule and any other portions of t-PA otherwise nativeto the source of the t-PA. It will be understood that natural allelicvariations exist and can occur among individuals, as demonstrated by oneor more amino acid differences in the amino acid sequence of t-PA ofeach individual.

The terms "wild-type t-PA" and "native t-PA" refer to native-sequencehuman t-PA, i.e., that encoded by the cDNA sequence reported in U.S.Pat. No. 4,766,075, issued Aug. 23, 1988, the disclosure of which isincorporated by reference. Amino acid site numbers or positions in thet-PA molecule are labelled in accordance with U.S. Pat. No. 4,766, 075,supra. The t-PA may be from any native source and includes thecorresponding proteins of various animals such as humans. In addition,the t-PA may be obtained from any recombinant expression system,including, for example, Chinese hamster ovary (CHO cells) or humanembryonic kidney 293 cells.

The terms "amino acid" and "amino acids" refer to all naturallyoccurring L-α-amino acids. The amino acids are identified by either thesingle-letter or three-letter designations:

    ______________________________________                                        Asp  D       aspartic acid                                                                              Ile  I     isoleucine                               Thr  T       threonine    Leu  L     leucine                                  Ser  S       serine       Tyr  Y     tyrosine                                 Glu  E       glutamic acid                                                                              Phe  F     phenylalanine                            Pro  P       proline      His  H     histidine                                Gly  G       glycine      Lys  K     lysine                                   Ala  A       alanine      Arg  R     arginine                                 Cys  C       cysteine     Trp  W     tryptophan                               Val  V       valine       Gln  Q     glutamine                                Met  M       methionine   Asn  N     asparagine                               ______________________________________                                    

These amino acids may be classified according to the chemicalcomposition and properties of their side chains. They are broadlyclassified into two groups, charged and uncharged. Each of these groupsis divided into subgroups to classify the amino acids more accurately:

I. Charged Amino Acids

Acidic Residues: aspartic acid, glutamic acid

Basic Residues: lysine, arginine, histidine

II. Uncharged Amino Acids

Hydrophilic Residues: serine, threonine, asparagine, glutamine

Aliphatic Residues: glycine, alanine, valine, leucine, isoleucine

Non-polar Residues: cysteine, methionine, proline

Aromatic Residues: phenylalanine, tyrosine, tryptophan

The terms "alteration", "amino acid sequence alteration", "variant" and"amino acid sequence variant" refer to t-PA molecules with somedifferences in their amino acid sequences as compared to native t-PA.Ordinarily, the variants will possess at least 80% homology with nativet-PA, and preferably, they will be at least about 90% homologous withnative t-PA. The amino acid sequence variants of t-PA falling withinthis invention possess substitutions, deletions, and/or insertions atcertain positions. These positions have been identified by the inventorsto be influential in modulating the clearance rate of t-PA.

Substitutional t-PA variants are those that have at least one amino acidresidue in the native t-PA sequence removed and a different amino acidinserted in its place at the same position. The substitutions may besingle, where only one amino acid in the molecule has been substituted,or they may be multiple, where two or more amino acids have beensubstituted in the same molecule.

Substantial changes in the activity of the t-PA molecule may be obtainedby substituting an amino acid with a side chain that is significantlydifferent in charge and/or structure from that of the native amino acid.This type of substitution would be expected to affect the structure ofthe polypeptide backbone and/or the charge or hydrophobicity of themolecule in the area of the substitution.

Moderate changes in the activity of the t-PA molecule would be expectedby substituting an amino acid with a side chain that is similar incharge and/or structure to that of the native molecule. This type ofsubstitution, referred to as a conservative substitution, would not beexpected to substantially alter either the structure of the polypeptidebackbone or the charge or hydrophobicity of the molecule in the area ofthe substitution.

Insertional t-PA variants are those with one or more amino acidsinserted immediately adjacent to an amino acid at a particular positionin the native t-PA molecule. Immediately adjacent to an amino acid meansconnected to either the α-carboxy or α-amino functional group of theamino acid. The insertion may be one or more amino acids. Ordinarily,the insertion will consist of one or two conservative amino acids. Aminoacids similar in charge and/or structure to the amino acids adjacent tothe site of insertion are defined as conservative. Alternatively, thisinvention includes insertion of an amino acid with a charge and/orstructure that is substantially different from the amino acids adjacentto the site of insertion.

Deletional variants are those with one or more amino acids in the nativet-PA molecule removed. Ordinarily, deletional variants will have one ortwo amino acids deleted in a particular region of the t-PA molecule.

The notations used throughout this application to describe t-PA aminoacid sequence variants are described below. The location of a particularamino acid in the polypeptide chain of t-PA is identified by a number.The number refers to the amino acid position in the amino acid sequenceof the mature, wild-type human t-PA polypeptide as disclosed in U.S.Pat. No. 4,766,075, issued Aug. 23, 1988. In the present application,similarly positioned residues in t-PA variants are designated by thesenumbers even though the actual residue number is not so numbered due todeletions or insertions in the molecule. This will occur, for example,with site-directed deletional or insertional variants. The amino acidsare identified using the one-letter code. Substituted amino acids aredesignated by identifying the wild-type amino acid on the left side ofthe number denoting the position in the polypeptide chain of that aminoacid, and identifying the substituted amino acid on the right side ofthe number.

For example, replacing the amino acid glutamic acid (E) at position 94of t-PA with alanine (A) is designated as E94A. Replacing glutamic acid(E) at position 94 with alanine and aspartic acid (D) at position 95with alanine would be indicated as E94A,D95A. Deletional variants areidentified by indicating the amino acid residue and position at eitherend of the deletion, inclusive, and placing the Greek letter delta, "Δ",to the left of the indicated amino acids. For example, a t-PA variantcontaining a deletion of amino acids 100-101 would be indicated asΔY100-R101, where Y and R indicate the amino acids tyrosine andarginine, respectively. Deletion of a single amino acid, for exampleY100, would be indicated as ΔY100. Insertional t-PA variants aredesignated by the use of brackets "[]" around the inserted amino acids,and the location of the insertion is denoted by indicating the positionof the amino acid on either side of the insertion. For example, aninsertion of the amino acids alanine (A) and valine (V) between glutamicacid at position 94 and aspartic acid at position 95 is indicated asE94[A,V]D95. For ease of reading, a comma "," is used to separatemultiple mutations that occur in a single molecule, and a semi-colon ";"is used to separate individual t-PA variant molecules that have beenconstructed, where several t-PA variant molecules are listed together.

The terms "clearance rate" and "clearance" refer to rate at which thet-PA molecule is removed from the bloodstream. Clearance is measuredwith respect to native t-PA, such that decreased clearance indicatesthat the t-PA variant is cleared more slowly than native t-PA, andincreased clearance indicates that the t-PA variant is cleared morerapidly than native t-PA.

The terms "biological activity", "biologically active", "activity" and"active" refer to the ability of the t-PA molecule to convertplasminogen to plasmin as measured in the S-2251 assay in the presenceof a plasma clot or in the presence of fibrin, the S-2288 assay, theplasma clot lysis assay, or other appropriate assays. The t-PA moleculemay be assayed in its one or two chain form, and the assay(s) may beconducted in the presence or absence of potential modulators of activitysuch as fibrin, fibrinogen, plasma and/or plasma clots.

The terms "DNA sequence encoding", "DNA encoding" and "nucleic acidencoding" refer to the order or sequence of deoxyribonucleotides along astrand of deoxyribonucleic acid. The order of these deoxyribonucleotidesdetermines the order of amino acids along the polypeptide chain. The DNAsequence thus codes for the amino acid sequence.

The terms "replicable expression vector" and "expression vector" referto a piece of DNA, usually double-stranded, which may have inserted intoit a piece of foreign DNA. Foreign DNA is defined as heterologous DNA,which is DNA not naturally found in the host cell. The vector is used totransport the foreign or heterologous DNA into a suitable host cell.Once in the host cell, the vector can replicate independently of thehost chromosomal DNA, and several copies of the vector and its inserted(foreign) DNA may be generated. In addition, the vector contains thenecessary elements that permit translating the foreign DNA into apolypeptide. Many molecules of the polypeptide encoded by the foreignDNA can thus be rapidly synthesized.

The terms "transformed host cell" and "transformed" refer to theintroduction of DNA into a cell. The cell is termed a "host cell", andit may be a prokaryotic or a eukaryotic cell. Typical prokaryotic hostcells include various strains of E. coli. Typical eukaryotic host cellsare mammalian, such as Chinese hamster ovary cells or human embryonickidney 293 cells. The introduced DNA is usually in the form of a vectorcontaining an inserted piece of DNA. The introduced DNA sequence may befrom the same species as the host cell or a different species from thehost cell, or it may be a hybrid DNA sequence, containing some foreignand some homologous DNA.

II. GENERAL METHODS A. Selection Of Variants

The clearance rate of t-PA can be modulated by altering the amino acidsequence of the molecule. The alterations may be insertions, deletionsand/or substitutions of amino acids. Preferably, the alterations will beby substitution of at least one amino acid in one or more regions of themolecule. A suitable technique for selecting the amino acid(s) to besubstituted is that of alanine-scanning mutagenesis, as described byCunningham and Wells (Science, 244: 1081 [1989]). In this technique, oneor more amino acids with charged side-chains are replaced by amino acidswith uncharged side-chains. Such changes are believed to affect theinteraction of the polypeptide with the surrounding aqueous environment.

For alanine-scanning mutagenesis, the amino acids for use insubstitution are those that will neutralize the charge of thecorresponding amino acid of wild-type t-PA. Any hydrophobic, aliphatic,aromatic, or non-polar amino acid can be used. Among these, amino acidswith small side-chains such as alanine, serine and threonine arepreferred to those with larger side chains such as valine, leucine, andisoleucine. Preferably, the amino acid used for replacement is eitherglycine, alanine, serine, threonine, glutamine, or asparagine. Mostpreferably, the amino acid used for replacement is alanine, or, atcertain positions, asparagine, glycine or glutamine. Alanine is the mostpreferred amino acid for this purpose because it eliminates theside-chain beyond the beta-carbon, and is thus less likely to alter thetertiary conformation of wild-type t-PA. Further, alanine is frequentlyfound in both buried and exposed regions of proteins (Chothia, J. Mol.Biol., 50:1 [1976]).

Exemplary variants with decreased clearance and/or increased half-lifewill have at least one alteration (amino acid substitution, deletionand/or insertion) of the wild-type t-PA amino acid sequence in eitherthe kringle-1 domain or the kringle-2 domain. The variants may alsocontain additional alterations of residues in other domains of thewild-type sequence that further enhance the properties of the molecule.For example, these additional alterations may serve to increasefibrin-binding specificity, specific activity, and/or zymogenicity ofthe t-PA variant.

In one preferred embodiment, the amino acid at position 94 or 95, or theamino acids at positions 236, 238, and 240, or combinations of thesepositions will be substituted, preferably with alanine, or glycine. Eachof these molecules may also contain other amino acid substitutions,deletions or insertions, preferably a substitution of asparagine forthreonine at position 103 (see WO 89/11531 published Nov. 30, 1989 andU.S. Ser. No. 07/480691 filed Feb. 15, 1990 now abandoned) and/or asubstitution of alanine or serine, or preferably glutamine, forasparagine at position 117. Representative t-PA variants of thisinvention include E94A; D95A; D95G; E94A,D95A; D236A,D238A,K240A;E94A,D95A,N117Q; E94A,D95A,D236A,D238A,K240A; T103N,D236A,D238A,K240A;and N117Q,D236A,D238A,K240A.

In addition, the molecules of this invention may be substituted ordeleted at certain positions to confer additional desired propertiesincluding increased fibrin specificity or zymogenicity. These positionsinclude, for example, deletion of amino acids 92 to 179, deletions inthe region of amino acids 174-261, modification at glycosylation sitessuch as position 184 and/or modifications in the region of amino acids244-255. Other key positions for modification are located throughout theprotease domain and include, for example, position 275 (see EPO 233, 013published Aug. 19, 1987 and WO 87/04722, published Aug. 13, 1987),position 277 (see EPO 297,066, published Dec. 28, 1988 and EPO 201,153published Nov. 12, 1986) and positions 296-299 as disclosed in WO90/02798 published Mar. 22, 1990.

B. Construction Of Variants

The t-PA amino acid sequence variants of this invention are preferablyconstructed by mutating the DNA sequence that encodes wild-type t-PA.Generally, particular regions or sites of the DNA will be targeted formutagenesis, and thus the general methodology employed to accomplishthis is termed site-directed mutagenesis. The mutations are made usingDNA modifying enzymes such as restriction endonucleases (which cleaveDNA at particular locations), nucleases (which degrade DNA) and/orpolymerases (which synthesize DNA).

1. Simple Deletions and Insertions

Restriction endonuclease digestion of DNA followed by ligation may beused to generate deletions, as described in section 15.3 of Sambrook etal. (Molecular Cloning: A Laboratory Manual, second edition, Cold SpringHarbor Laboratory Press, New York [1989]). To use this method, it ispreferable that the foreign DNA be inserted into a plasmid vector. Arestriction map of both the foreign (inserted) DNA and the vector DNAmust be available, or the sequence of the foreign DNA and the vector DNAmust be known. The foreign DNA must have unique restriction sites thatare not present in the vector. Deletions are then made in the foreignDNA by digesting it between these unique restriction sites, using theappropriate restriction endonucleases under conditions suggested by themanufacturer of the enzymes. If the restriction enzymes used createblunt ends or compatible ends, the ends can be directly ligated togetherusing a ligase such as bacteriophage T4 DNA ligase and incubating themixture at 16° C. for 1-4 hours in the presence of ATP and ligase bufferas described in section 1.68 of Sambrook et al., supra. If the ends arenot compatible, they must first be made blunt by using the Klenowfragment of DNA polymerase I or bacteriophage T4 DNA polymerase, both ofwhich require the four deoxyribonucleotide triphosphates to fill-in theoverhanging single-stranded ends of the digested DNA. Alternatively, theends may be blunted using a nuclease such as nuclease S1 or mung-beannuclease, both of which function by cutting back the overhanging singlestrands of DNA. The DNA is then religated using a ligase. The resultingmolecule is a t-PA deletion variant.

A similar strategy may be used to construct insertion variants, asdescribed in section 15.3 of Sambrook et al., supra. After digestion ofthe foreign DNA at the unique restriction site(s), an oligonucleotide isligated into the site where the foreign DNA has been cut. Theoligonucleotide is designed to code for the desired amino acids to beinserted and additionally has 5' and 3' ends that are compatible withthe ends of the foreign DNA that have been digested, such that directligation is possible.

2. Oligonucleotide-Mediated Mutagenesis

Oligonucleotide-directed mutagenesis is the preferred method forpreparing the substitution variants of this invention. It may also beused to conveniently prepare the deletion and insertion variants of thisinvention. This technique is well known in the art as described byAdelman et al. (DNA, 2:183 [1983]).

Generally, oligonucleotides of at least 25 nucleotides in length areused to insert, delete or substitute two or more nucleotides in the t-PAmolecule. An optimal oligonucleotide will have 12 to 15 perfectlymatched nucleotides on either side of the nucleotides coding for themutation. This ensures that the oligonucleotide will hybridize properlyto the single-stranded DNA template molecule. The oligonucleotides arereadily synthesized using techniques well known in the art such as thatdescribed by Crea et al. (Proc. Nat'l. Acad. Sci. USA, 75:5765 [1978]),specifically incorporated by reference.

The DNA template molecule is the single-stranded form of the vector withits wild-type cDNA t-PA insert. The single-stranded template can only begenerated by those vectors that are either derived from bacteriophageM13 vectors (the commercially available M13mp18 and M13mp19 vectors aresuitable), or those vectors that contain a single-stranded phage originof replication as described by Veira et al. (Meth. Enzymol., 153:3[1987]). Thus, the cDNA t-PA that is to be mutated must be inserted intoone of these vectors in order to generate single-stranded template.Production of the single-stranded template is described in sections4.21-4.41 of Sambrook et al., supra.

To mutagenize the wild-type t-PA, the oligonucleotide is annealed to thesingle-stranded DNA template molecule under suitable hybridizationconditions. A DNA polymerizing enzyme, usually the Klenow fragment of E.coli DNA polymerase I, is then added. This enzyme uses theoligonucleotide as a primer to complete the synthesis of themutation-bearing strand of DNA. Thus, a heteroduplex molecule is formedsuch that one strand of DNA encodes the wild-type t-PA inserted in thevector, and the second strand of DNA encodes the mutated form of t-PAinserted into the same vector. This heteroduplex molecule is thentransformed into a suitable host cell, usually a prokaryote such as E.coli JM101. After growing the cells, they are plated on to agaroseplates and screened using the oligonucleotide primer radiolabeled with32-P to identify the colonies that contain the mutated t-PA. Thesecolonies are selected, and the DNA is sequenced to confirm the presenceof mutations in the t-PA molecule.

Mutants with more than one amino acid substituted may be generated inone of several ways. If the amino acids are located close together inthe polypeptide chain, they may be mutated simultaneously using oneoligonucleotide that codes for all of the desired amino acidsubstitutions. If however, the amino acids are located some distancefrom each other (separated by more than ten amino acids, for example) itis more difficult to generate a single oligonucleotide that encodes allof the desired changes. Instead, one of two alternative methods may beemployed. In the first method, a separate oligonucleotide is generatedfor each amino acid to be substituted. The oligonucleotides are thenannealed to the single-stranded template DNA simultaneously, and thesecond strand of DNA that is synthesized from the template will encodeall of the desired amino acid substitutions. The alternative methodinvolves two or more rounds of mutagenesis to produce the desiredmutant. The first round is as described for the single mutants:wild-type t-PA DNA is used for the template, an oligonucleotide encodingthe first desired amino acid substitution(s) is annealed to thistemplate, and the heteroduplex DNA molecule is then generated. Thesecond round of mutagenesis utilizes the mutated DNA produced in thefirst round of mutagenesis as the template. Thus, this template alreadycontains one or more mutations. The oligonucleotide encoding theadditional desired amino acid substitution(s) is then annealed to thistemplate, and the resulting strand of DNA now encodes mutations fromboth the first and second rounds of mutagenesis. This resultant DNA canbe used as a template in a third round of mutagenesis, and so on.

To express the DNA encoding the t-PA variant as a polypeptide, this DNAis excised from the vector and inserted into an expression vector thatis appropriate for eukaryotic host cell expression. Chinese hamsterovary (CHO) cells are preferred for long-term stable t-PA production.However, this invention is not limited to expression of t-PA variants inCHO cells, as it is known that numerous other cell types can be used,particularly if only transient expression of the t-PA variants isnecessary, as for experimental purposes.

C. Host Cell Cultures And Vectors 1. Prokaryotic Cells

Prokaryotes are the preferred host cells for the initial cloning stepsof this invention. They are particularly useful for rapid production oflarge amounts of DNA, for production of single-stranded DNA templatesused for site-directed mutagenesis, for screening many mutantssimultaneously, and for DNA sequencing of the mutants generated.Suitable prokaryotic host cells include E. coli K12 strain 294 (ATCCnumber 31,446), E. coli strain W3110 (ATCC number 27,325) E. coli X1776(ATCC number 31,537), and E. coli B; however many other strains of E.coli, such as HB101, JM101, NM522, NM538, NM539, and many other speciesand genera of prokaryotes may be used as well.

Prokaryotes may also be used as hosts for expression of DNA sequences.The E. coli strains listed above, bacilli such as Bacillus subtilis,other enterobacteriaceae such as Salmonella typhimurium or Serratiamarcesans, and various Pseudomonas species may all be used as hosts.

Plasmid vectors containing replicon and control sequences that arederived from species compatible with the host cell are used with thesehosts. The vector usually has a replication site, marker genes thatprovide phenotypic selection in transformed cells, one or morepromoters, and a polylinker region containing several restriction sitesfor insertion of foreign DNA. Plasmids typically used for transformationof E. coli include pBR322, pUC18, pUC19, pUCI18, pUC119, and BluescriptM13, all of which are described in sections 1.12-1.20 of Sambrook etal., supra. However, many other suitable vectors are available as well.These vectors contain genes coding for ampicillin and/or tetracyclineresistance which enables cells transformed with these vectors to grow inthe presence of these antibiotics.

The promoters most commonly used in prokaryotic vectors include theβ-lactamase (penicillinase) and lactose promoter systems (Chang et al.Nature, 375:615 [1978]; Itakura et al., Science, 198:1056 [1977];Goeddel et al., Nature, 281:544 [1979]) and a tryptophan (trp) promotersystem (Goeddel et al., Nucl. Acids Res., 8:4057 [1980]; EPO Appl. Publ.No. 36,776), and the alkaline phosphatase systems. While these are themost commonly used, other microbial promoters have been utilized, anddetails concerning their nucleotide sequences have been published,enabling a skilled worker to ligate them functionally into plasmidvectors (see Siebenlist et al., Cell, 20:269 [1980]).

2. Eukaryotic Microbes

Eukaryotic microbes such as yeasts may be used to practice thisinvention. The baker's yeast Saccharomyces cerevisiae, is a commonlyused eukaryotic microorganism, although several other strains areavailable. The plasmid YRp7 (Stinchcomb et al., Nature, 282:39 [1979];Kingsman et al., Gene, 7:141 [1979]; Tschemper et al., Gene, 10:157[1980]) is commonly used as an expression vector in Saccharomyces. Thisplasmid contains the trp1 gene that provides a selection marker for amutant strain of yeast lacking the ability to grow in tryptophan, suchas strains ATCC No. 44,076 and PEP4-1 (Jones, Genetics, 85:12 [1977]).The presence of the trp1 lesion as a characteristic of the yeast hostcell genome then provides an effective environment for detectingtransformation by growth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem., 255:2073[1980]) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg.,7:149 [1968]; Holland et al., Biochemistry, 17: 4900 [1978]), such asenolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. In the construction ofsuitable expression plasmids, the termination sequences associated withthese genes are also ligated into the expression vector 3' of thesequence desired to be expressed to provide polyadenylation of the mRNAand termination. Other promoters that have the additional advantage oftranscription controlled by growth conditions are the promoter regionfor alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,degradative enzymes associated with nitrogen metabolism, and theaforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization. Any plasmid vectorcontaining yeast-compatible promoter, origin of replication andtermination sequences is suitable.

3. Eukaryotic Multicellular Organisms

Cell cultures derived from multicellular organisms may be used as hoststo practice this invention. While both invertebrate and vertebrate cellcultures are acceptable, vertebrate cell cultures, particularlymammalian cultures, are preferable. Examples of suitable cell linesinclude monkey kidney CVI line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney line 293S (Graham et al., J. Gen. Virol..36:59 [1977]); baby hamster kidney cells (BHK, ATCC CCL 10); Chinesehamster ovary cells (Urlab and Chasin, Proc. Natl. Acad. Sci USA,77:4216 [1980]); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243[1980]); monkey kidney cells (CVI-76, ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442 ; human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor cells (MMT060562, ATCC CCL 51); rat hepatoma cells (HTC, MI.54, Baumann et al., J.Cell Biol., 85:1 [1980]); and TRI cells (Mather et al., Annals N.Y.Acad. Sci., 383:44 [1982]). Expression vectors for these cellsordinarily include (if necessary) DNA sequences for an origin ofreplication, a promoter located in front of the gene to be expressed, aribosome binding site, an RNA splice site, a polyadenylation site, and atranscription terminator site.

Promoters used in mammalian expression vectors are often of viralorigin. These viral promoters are commonly derived from polyoma virus,Adenovirus2, and most frequently Simian Virus 40 (SV40). The SV40 viruscontains two promoters that are termed the early and late promoters.These promoters are particularly useful because they ar both easilyobtained from the virus as one DNA fragment that also contains the viralorigin of replication (Fiers et al., Nature, 273:113 [1978]). Smaller orlarger SV40 DNA fragments may also used, provided they contain theapproximately 250-bp sequence extending from the HindIII site toward theBglI site located in the viral origin of replication.

Alternatively, promoters that are naturally associated with the foreigngene (homologous promoters) may be used provided that they arecompatible with the host cell line selected for transformation.

An origin of replication may be obtained from an exogenous source, suchas SV40 or other virus (e.g., Polyoma, Adeno, VSV, BPV) and insertedinto the cloning vector. Alternatively, the origin of replication may beprovided by the host cell chromosomal replication mechanism. If thevector containing the foreign gene is integrated into the host cellchromosome, the latter is often sufficient.

Satisfactory amounts of human t-PA are produced by transformed cellcultures. However, the use of a secondary DNA coding sequence canenhance production levels. The secondary coding sequence typicallycomprises the enzyme dihydrofolate reductase (DHFR). The wild-type formof DHFR is normally inhibited by the chemical methotrexate (MTX). Thelevel of DHFR expression in a cell will vary depending on the amount ofMTX added to the cultured host cells. An additional feature of DHFR thatmakes it particularly useful as a secondary sequence is that it can beused as a selection marker to identify transformed cells.

Two forms of DHFR are available for use as secondary sequences,wild-type DHFR and MTX-resistant DHFR. The type of DHFR used in aparticular host cell depends on whether the host cell is DHFR deficient(such that it either produces very low levels of DHFR endogenously, orit does not produce functional DHFR at all). DHFR-deficient cell linessuch as the CHO cell line described by Urlaub and Chasin (Proc. Natl.Acad. Sci. (USA) 77:4216 [1980]) are transformed with wild-type DHFRcoding sequences. After transformation, these DHFR-deficient cell linesexpress functional DHFR and are capable of growing in a culture mediumlacking the nutrients hypoxanthine, glycine and thymidine.Nontransformed cells will not survive in this medium.

The MTX-resistant form of DHFR can be used as a means of selecting fortransformed host cells in those host cells that endogenously producenormal amounts of functional DHFR that is MTX sensitive. The CHO-Kl cellline (ATCC number CL 61) possesses these characteristics, and is thus auseful cell line for this purpose. The addition of MTX to the cellculture medium will permit only those cells transformed with the DNAencoding the MTX-resistant DHFR to grow. The nontransformed cells willbe unable to survive in this medium.

4. Secretion Systems

Many eukaryotic proteins normally secreted from the cell contain anendogenous signal sequence as part of the amino acid sequence. Thissequence targets the protein for export from the cell via theendoplasmic reticulum and Golgi apparatus. The signal sequence istypically located at the amino terminus of the protein, and ranges inlength from about 13 to about 36 amino acids. Although the actualsequence varies among proteins, all known eukaryotic signal sequencescontain at least one positively charged residue and a highly hydrophobicstretch of 10-15 amino acids (usually rich in the amino acids luecine,isoleucine, alanine, valine and phenylalanine) near the center of thesignal sequence. The signal sequence is normally absent from thesecreted form of the protein, as it is cleaved by a signal peptidaselocated on the endoplasmic reticulum during translocation of the proteininto the endoplasmic reticulum. The protein with its signal sequencestill attached is often referred to as the `pre-protein` or the immatureform of the protein.

However, not all secreted proteins contain an amino terminal signalsequence that is cleaved. Some proteins, such as ovalbumin, contain asignal sequence that is located on an internal region of the protein.This sequence is not normally cleaved during translocation.

Proteins normally found in the cytoplasm can be targeted for secretionby linking a signal sequence to the protein. This is readilyaccomplished by ligating DNA encoding a signal sequence to the 5' end ofthe DNA encoding the protein and then expressing this fusion protein inan appropriate host cell. The DNA encoding the signal sequence may beobtained as a restriction fragment from any gene encoding a protein witha signal sequence. Thus, prokaryotic, yeast, and eukaryotic signalsequences may be used herein, depending on the type of host cellutilized to practice the invention. The DNA encoding the signal sequenceportion of the gene is excised using appropriate restrictionendonucleases and then ligated to the DNA encoding the protein to besecreted, i.e. t-PA.

Selection of a functional signal sequence requires that the signalsequence is recognized by the host cell signal peptidase such thatcleavage of that signal sequence and secretion of the protein willoccur. The DNA and amino acid sequence encoding the signal sequenceportion of several eukaryotic genes including, for example, human growthhormone, proinsulin, and proalbumin are known (see Stryer, Biochemistry,W.H. Freeman and Company, New York [1988], p. 769) and can be used assignal sequences in appropriate eukaryotic host cells. Yeast signalsequences, as for example acid phosphatase (Arima et al., Nuc. AcidsRes., 11:1657 [1983]), alpha-factor, alkaline phosphatase and invertasemay be used to direct secretion from yeast host cells. Prokaryoticsignal sequences from genes encoding, for example, LamB or OmpF (Wong etal., Gene 68:193 1988]), MalE, PhoA, or beta-lactamase, as well as othergenes, may be used to target proteins from prokaryotic cells into theculture medium.

An alternative technique to provide a protein of interest with a signalsequence such that it may be secreted is to chemically synthesize theDNA encoding the signal sequence. In this method, both strands of anoligonucleotide encoding the selected signal sequence are chemicallysynthesized and then annealed to each other to form a duplex. Thedouble-stranded oligonucleotide is then ligated to the 5' end of the DNAencoding the protein.

The construct containing the DNA encoding the protein with the signalsequence ligated to it can then be ligated into a suitable expressionvector. This expression vector is transformed into a an appropriate hostcell and the protein of interest is expressed and secreted.

D. Transformation Methods

Cultures of mammalian host cells and other host cells that do not haverigid cell membrane barriers are usually transformed using the calciumphosphate method as originally described by Graham and Van der Eb(Virology, 52:546 [1978]) and modified as described in sections16.32-16.37 of Sambrook et al. supra. However, other methods forintroducing DNA into cells such as Polybrene (Kawai and Nishizawa, Mol.Cell. Biol., 4:1172 [1984]), protoplast fusion (Schaffner, Proc. Natl.Acad. Sci. USA, 77:2163 [1980]), electroporation (Neumann et al., EMBOJ., 1:841 [1982]), and direct microinjection into nuclei (Capecchi,Cell, 22:479 [1980]) may also be used.

Yeast host cells are generally transformed using the polyethylene glycolmethod, as described by Hinnen (Proc. Natl. Acad. Sci. U.S.A., 75:1929[1978]).

Prokaryotic host cells or other host cells with rigid cell walls arepreferably transformed using the calcium chloride method as described insection 1.82 of Sambrook et al., supra. Alternatively, electroporationmay be used for transformation of these cells.

E. Cloning Methods

Construction of suitable vectors containing DNA encoding replicationsequences, regulatory sequences, phenotypic selection genes and theforeign DNA of interest are prepared using standard recombinant DNAprocedures. Isolated plasmids and DNA fragments are cleaved, tailored,and ligated together in a specific order to generate the desiredvectors.

The DNA is cleaved using the appropriate restriction enzyme or enzymesin a suitable buffer. In general, about 0.2-1 μg of plasmid or DNAfragments is used with about 1-2 units of the appropriate restrictionenzyme in about 20 μl of buffer solution. (Appropriate buffers, DNAconcentrations, and incubation times and temperatures are specified bythe manufacturers of the restriction enzymes.) Generally, incubationtimes of about one or two hours at 37° C. are adequate, although severalenzymes require higher temperatures. After incubation, the enzymes andother contaminants are removed by extraction of the digestion solutionwith a mixture of phenol and chloroform, and the DNA is recovered fromthe aqueous fraction by precipitation with ethanol.

To ligate the DNA fragments together to form a functional vector, theends of the DNA fragments must be compatible with each other. In somecases the ends will be directly compatible after endonuclease digestion.However, it may be necessary to first convert the sticky ends, commonlyproduced by endonuclease digestion, to blunt ends to make themcompatible for ligation. To blunt the ends, the DNA is treated in asuitable buffer for at least 15 minutes at 15° C. with 10 units of theKlenow fragment of DNA Polymerase I (Klenow) in the presence of the fourdeoxynucleotide triphosphates. It is then purified by phenolchloroformextraction and ethanol precipitation.

The cleaved DNA fragments may be size-separated and selected using DNAgel electrophoresis. The DNA may be electrophoresed through either anagarose or a polyacrylamide matrix. The selection of the matrix willdepend on the size of the DNA fragments to be separated. Afterelectrophoresis, the DNA is extracted from the matrix by electroelution,or, if low-melting agarose has been used as the matrix, by melting theagarose and extracting the DNA from it, as described in sections6.30-6.33 of Sambrook et al., supra.

The DNA fragments that are to be ligated together (previously digestedwith the appropriate restriction enzymes such that the ends of eachfragment to be ligated are compatible) are present in solution in aboutequimolar amounts. The solution will also contain ATP, ligase buffer anda ligase such as T4 DNA ligase at about 10 units per 0.5 μg of DNA. Ifthe DNA fragment is to be ligated into a vector, the vector is firstlinearized by cutting with the appropriate restriction endonuclease(s)and then phosphatased with either bacterial alkaline phosphatase or calfintestinal alkaline phosphatase. This prevents self-ligation of thevector during the ligation step.

After ligation, the vector with the foreign gene now inserted istransformed into a suitable host cell, most commonly a prokaryote suchas E. coli K12 strain 294 (ATCC number 31,446) or another suitable E.coli strain. The transformed cells are selected by growth on anantibiotic, commonly tetracycline (tet) or ampicillin (amp), to whichthey are rendered resistant due to the presence of tet and/or ampresistance genes on the vector. If the ligation mixture has beentransformed into a eukaryotic host cell, transformed cells may beselected by the DHFR/MTX system described above. The transformed cellsare grown in culture and the plasmid DNA (plasmid refers to the vectorligated to the foreign gene of interest) is then isolated. This plasmidDNA is then analyzed by restriction mapping and/or DNA sequencing. DNAsequencing is generally performed by either the method of Messing etal., Nucleic Acids Res., 9:309 (1981) or by the method of Maxam et al.,Methods of Enzymology, 65:499 (1980).

After mammalian host cells have been stably transformed with the DNA,the DHFR-protein-coding sequences are amplified by growing the host cellcultures in the presence of approximately 200-500 nM of methotrexate.The effective range of concentrations of MTX is highly dependent uponthe nature of the DHFR gene and protein and the characteristics of thehost. Clearly, generally defined upper and lower limits cannot beascertained. Suitable concentrations of other folic acid analogs orother compounds that inhibit DHFR may also be used. MTX itself is,however, convenient, readily available, and effective.

As discussed above, t-PA variants are preferably produced by means ofmutation(s) that are generated using the method of site-specificmutagenesis. This method requires the synthesis and use of specificoligonucleotides that encode both the sequence of the desired mutationand a sufficient number of adjacent nucleotides to allow theoligonucleotide to stably hybridize to the DNA template.

F. Pharmaceutical Compositions

The compounds of the present invention can be formulated according toknown methods to prepare pharmaceutically useful compositions, wherebythe t-PA product is combined in admixture with a pharmaceuticallyacceptable carrier. Suitable carriers and their formulations aredescribed in Remington's Pharmaceutical Sciences, 16th ed., 1980, MackPublishing Co., edited by Oslo et al., specifically incorporated byreference. These compositions will typically contain an effective amountof the t-PA variant, for example, from on the order of about 0.5 toabout 5 mg/ml, together with a suitable amount of carrier to preparepharmaceutically acceptable compositions suitable for effectiveadministration to the patient. The t-PA variant may be administeredparenterally to patients suffering from cardiovascular diseases orconditions, or by other methods that ensure its delivery to thebloodstream in an effective form.

Compositions particularly well suited for the clinical administration ofthe t-PA variants used to practice this invention include sterileaqueous solutions or sterile hydratable powders such as lyophilizedprotein. Typically, an appropriate amount of a pharmaceuticallyacceptable salt is also used in the formulation to render theformulation isotonic. A buffer such as arginine base in combination withphosphoric acid is also typically included at an appropriateconcentration to maintain a suitable pH, generally from 5.5 to 7.5. Inaddition, a compound such as glycerol may be included in the formulationto help maintain the shelf-life.

Dosages and desired drug concentrations of pharmaceutical compositionsof this invention may vary depending on the particular use envisioned.For example, in the treatment of deep vein thrombosis or peripheralvascular disease, "bolus" doses, on the order of about 0.05 to about 0.2mg/kg, will typically be preferred with subsequent administrations of onthe order of about 0.1 to about 0.2 mg/kg administered to maintain afairly constant blood level, preferably of on the order of about 3μg/ml.

However, for use in connection with emergency medical care facilitieswhere infusion capability is generally not available and due to thegenerally critical nature of the underlying disease (e.g., embolism,infarct), it is usually desirable to provide larger initial doses, suchas an intravenous bolus of on the order of about 0.3 mg/kg.

For example, the t-PA variant is suitably administered parenterally tosubjects suffering from cardiovascular diseases or conditions. Dosageand dose rate may be parallel to or higher than that currently in use inclinical investigations of other cardiovascular, thrombolytic agents,e.g., about 1-2 mg/kg body weight as an intravenous or intra-arterialdose over 1.5 to 12 hours in human patients suffering from myocardialinfarction, pulmonary embolism, etc.

As one example of an appropriate dosage form, a vial containing 50 mgt-PA, arginine, phosphoric acid, and polysorbate 80 is reconstitutedwith 50 ml sterile water for injection and mixed with a suitable volumeof 0.9 percent sodium chloride injection.

The t-PA variants of this invention are also useful for preventingfibrin deposition or adhesion formation or reformation. One embodimentof this use is described in EPO 297,860 published Jan. 4, 1989, thedisclosure of which is incorporated by reference. Generally, this typeof treatment involves topical administration of a composition to a siteof potential fibrin or adhesion formation wherein the compositioncomprises a therapeutically effective amount of the t-PA variant in asparingly soluble form that is continuously released at that site for aperiod of time of about from three days to two weeks. Typically, thet-PA variant is administered at a dosage sufficient to prevent fibrindeposition or formation of adhesions following surgery, infection,trauma, or inflammation. Usually, this amount is from 0.02 mg/g of gelto 25 mg/g of gel, with preferred amounts from 0.20 mg/g gel to about2.5 mg/g gel, most preferably from 0.25 mg/g gel to about 1.0 mg/g gel.

Each t-PA variant used to prevent adhesion formation and/or fibrindeposition is typically formulated in a semisolid, mucilagenous,pharmaceutically inert carrier for positioning the enzyme at the site ofpotential adhesion formation. The carrier includes long-chainhydrocarbons or vegetable oils and waxes composed of mixtures ofmodified saturated and unsaturated fatty acid glycerides or mixtures ofmodified saturated and unsaturated fatty acid glycerides. Examplesinclude semisolid vehicles such as petroleum jelly or semi-syntheticglycerides, polyhydroxy solvents such as glycerol, long-chainhydrocarbons, bioerodable polymers, or liposomes.

The decreased clearance rate of the t-PA variants of this invention mayrender them suitable for rapid intravenous injection, particularly as abolus, for example. This would simplify the method of administration oft-PA and might permit the use of t-PA in situations where medicalequipment is limited, such as in emergency vehicles staffed withparamedic personnel. In addition, the extended clearance rate of theset-PA variants may also permit administration of lower initial dosesand/or low-dose extended therapy that may be necessary to avoidreocclusion following acute thrombolysis, or for extended thrombolysisthat may be necessary in cases of peripheral vascular occlusion.

In order to simplify the examples certain commonly used methods arereferenced by the phrases below.

"Plasmids" are designated by a lower case p followed by an alphanumericdesignation. The starting plasmids used in this invention are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from such available plasmids using publishedprocedures. In addition, other equivalent plasmids are known in the artand will be apparent to the ordinary artisan.

"Digestion", "cutting" or "cleaving" of DNA refers to catalytic cleavageof the DNA with an enzyme that acts only at particular locations in theDNA. These enzymes are called restriction endonucleases, and the sitealong the DNA sequence where each enzyme cleaves is called a restriotionsite. The restriction enzymes used in this invention are commerciallyavailable and are used according to the instructions supplied by themanufacturers. Restriction enzymes are designated by abbreviationscomposed of a capital letter followed by two or three lower case lettersrepresenting the microorganism from which each restriction enzyme wasobtained. These letters are followed by one or more Roman numerals thatidentify the particular enzyme. In general, about 1 μg of plasmid or DNAfragment is used with about 2 units of enzyme in about 20 μl of buffersolution. The appropriate buffer, substrate concentration, incubationtemperature, and incubation time for each enzyme is specified by themanufacturer. After incubation, the enzyme and other contaminants areremoved from the DNA by extraction with a solution of phenol-chloroform,and the digested DNA is recovered from the aqueous fraction byprecipitation with ethanol. Digestion with a restriction enzyme may befollowed by treatment with bacterial alkaline phosphatase or calfintestinal alkaline phosphatase. This prevents the two restrictioncleaved ends of a DNA fragment from "circularizing" or forming a closedloop that would impede insertion of another DNA fragment at therestriction site. Unless otherwise stated, digestion of plasmids is notfollowed by 5' terminal dephosphorylation. These procedures and reagentsfor dephosphorylation are described in sections 1.60-1.61 and sections3.38-3.39 of Sambrook et al., supra.

"Recovery" or "isolation" of a given fragment of DNA from a restrictiondigest means separation of the resulting DNA fragment on apolyacrylamide or an agarose gel by electrophoresis, identification ofthe fragment of interest by comparison of its mobility versus that ofmarker DNA fragments of known molecular weight, removal of the gelsection containing the desired fragment, and separation of the gel from:DNA. This procedure is known generally. For example, see R. Lawn et al.,1981, Nucleic Acids Res. 9:6103-6114, and D. Goeddel et al., 1980,Nucleic Acids Res. 8:4057.

"Southern Analysis" is a method by which the presence of DNA sequencesin a digest or DNA-containing composition is confirmed by hybridizationto a known, labelled oligonucleotide or DNA fragment. Southern analysisrefers to the separation of digested DNA on an agarose gel, denaturationof the DNA, and transfer of the DNA from the gel to a nitrocellulose ornylon membrane using methods originally described by Southern (J. Mol.Biol., 98:503 [1975]) and modified as described in sections 9.31-9.57 ofSambrook et al., supra.

"Transformation" means introducing DNA into an organism so that the DNAis replicable, either as an extrachromosomal element or chromosomalintegrant. The method used for transformation depends on whether thehost cell is a eukaryote or a prokaryote. The method used to transformprokaryotes is the calcium chloride method as described in section 1.82of Sambrook et al., supra. Eukaryotes are transformed using the calciumphosphate method as described in sections 16.32-16.37 of Sambrook etal., supra.

"Ligation" refers to the process of forming phosphodiester bonds betweentwo double stranded DNA fragments using the enzyme ligase in a suitablebuffer that also contains ATP.

"Oligonucleotide" refers to short length single or double strandedsequences of deoxyribonucleotides linked via phosphodiester bonds. Theoligonucleotides are chemically synthesized by known methods andpurified on polyacrylamide gels.

The following examples merely illustrate the best mode now contemplatedfor practicing the invention, but should not be construed to limit theinvention. All literature citations herein are expressly incorporated byreference.

EXAMPLE I

A strategy known as alanine-scanning mutagenesis (ALA-scan), describedin Cunningham and Wells, supra, was used to construct the t-PA variants.This method involved the identification of small regions of the t-PAmolecule that contain charged amino acid side chains. Without limitationto any one theory, it is believed that either these regions containingclusters of charge, or neighboring regions, or both, are responsible forthe interaction of the t-PA molecule with its substrate and variousother compounds that may modulate its activity. Some of the chargedamino acids in each region (i.e., Arg, Asp, His, Lys, and Glu) werereplaced with alanine to assess the importance of the particular regionto the overall clearance rate of the t-PA molecule.

I. Construction of the Expression Vector pRK.t-PA

Plasmid pRK7 was used as the vector for generation of the t-PA mutants.pRK7 is identical to pRK5 (EP Publication Number 307,247 published Mar.15, 1989), except that the order of the endonuclease restriction sitesin the polylinker region between ClaI and HindIII is reversed. The t-PAcDNA (Pennica et al., Nature, 301:214 [1983]) was prepared for insertioninto the vector by cutting with restriction endonuclease HindIII (whichcuts 496 base pairs 5' of the ATG start codon) and restrictionendonuclease BalI (which cuts 276 base pairs downstream of the TGA stopcodon). This cDNA was ligated into pRK7 previously cut with HindIII andSmaI using standard ligation procedures as described in sections1.68-1.69 of Sambrook et al., supra. This construct was named pRK.t-PA.

II. Site Directed Mutagenesis of pRK7-t-PA

Site-directed mutagenesis of t-PA cDNA was performed by the method ofTaylor et al. (Nucl. Acids. Res., 13:8765 [1985]) using a kit purchasedfrom the Amersham Corporation (catalog number RPN 1253). For generationof the desired mutants, oligonucleotides of sequences coding for thedesired amino acid substitutions were synthesized and used as primers.These oligonucleotides were annealed to single-stranded pRK7-t-PA thathad been prepared by standard procedures (Viera et al., Meth. Enz.,143:3 [1987]).

A mixture of three deoxyribonucleotides, deoxyriboadenosine (dATP),deoxyriboguanosine (dGTP), and deoxyribothymidine (dTTP), was combinedwith a modified thio-deoxyribocytosine called dCTP(aS) provided in thekit by the manufacturer of the kit, and added to the single-strandedpRK7-t-PA to which was annealed the oligonucleotide.

Upon addition of DNA polymerase to this mixture, a strand of DNAidentical to pRK7-t-PA except for the mutated bases was generated. Inaddition, this new strand of DNA contained dCTP(aS) instead of dCTP,which served to protect it from restriction endonuclease digestion.After the template strand of the double-stranded heteroduplex was nickedwith an appropriate restriction enzyme, the template strand was digestedwith ExoIII nuclease past the region that contained the mutagenicoligomer. The reaction was then stopped to leave a molecule that wasonly partly single-stranded. A complete double-stranded DNA homoduplexmolecule was then formed by DNA polymerase in the presence of all fourdeoxyribonucleotide triphosphates, ATP, and DNA ligase.

The oligonucleotides listed in Table A were synthesized for use asprimers to generate the pRK7-t-PA variants, also listed in Table A,using the ALA-scan methodology described above:

                                      TABLE A                                     __________________________________________________________________________    Variant     Oligonucleotide                                                   __________________________________________________________________________    *E94A       5' GCCCTGGTCCGCGTAGCACGTGGC 3'                                    *D95A       5' GATGCCCTGCGCCTCGTAGCACGT 3'                                    *D95G       5' GATGCCCTGGCCCTCGTAGCACGT 3'                                    *E94A,D95A  5' GATGCCCTGGGCCGCGTAGCACGT 3'                                    *D236A,D238A,K240A                                                                        5' GGCACCAGGGCGCGGCAGCCCCAGCAGGATTCCG 3'                          T103N       5' TGTGCTCCAATTGCCCCTGTAGCT 3'                                    N117Q       5' CAACGCGCTGCTTTGCCAGTTGGT 3'                                    __________________________________________________________________________

The asterisks indicate variants that are illustrative of this invention.

Although the variants E94A,D95A and D236A,D238A,K240A are actuallymultiple mutants, as they contain more than one amino acid substitution,they were each generated using only one oligonucleotide. This waspossible since the substituted amino acids are located very close toeach other in the polypeptide chain.

A slight variation of the above described procedure was employed toprepare additional multiple mutants illustrative of this invention. Forthese mutants, described below, the template DNA was not wild-type t-PA(pRK7.t-PA). Instead, the templates used were those that contained atleast a single mutation, i.e., the DNA produced in construction of thesingle mutants listed above. The DNA used as the template, and theoligonucleotide used to generate the additional mutation(s) for eachmultiple mutant made is listed in Table B below. The DNA sequence ofeach oligonucleotide is set forth in Table A above.

                  TABLE B                                                         ______________________________________                                        Multiple Mutant                                                                            DNA Template Oligonucleotide                                     ______________________________________                                        *E94A,D95A,T103N                                                                           E94A,D95A    T103N                                               *E94A,D95A,N117Q                                                                           E94A,D95A    N117Q                                               *E94A,D95A,D236A,                                                                          D236A,D238A, E94A,E95A                                           D238A,K240A  K240A                                                            T103N,N117Q  T103N        N117Q                                               *T103N,D236A,                                                                              T103N        D236A,D238A,K240A                                   D238A,K240A                                                                   *N117Q,D236A,                                                                              N117Q        D236A,D238A,K240A                                   D238A,K240A                                                                   ______________________________________                                    

The asterisks indicate variants that are illustrative of this invention.

III. Bacterial Transformation and DNA Preparation

The mutant t-PA constructs generated using the protocol above weretransformed into E. coli host strain MM294tonA using the standardcalcium chloride procedure (sections 1.76-1.84 of Sambrook et al.,supra) for preparation and transformation of competent cells. The E.coli strain MM294tonA (which is resistant to T1 phage) was prepared bythe insertion and subsequent imprecise excision of a Tn10 transposoninto the tonA gene. This gene was then inserted, using transposoninsertion mutagenesis (Kleckner et al., J. Mol. Biol., 116: 125-159[1977]), into E. coli host MM294 (ATCC 31,446).

DNA was extracted from individual colonies of bacterial transformantsusing the standard miniprep procedure described in sections 1.25-1.31 ofSambrook et al., supra. The plasmids were further purified by passageover a Sephacryl CL6B spin column, and then analyzed by DNA sequencingand by restriction endonuclease digestion and agarose gelelectrophoresis.

IV. Transformation of Eukaryotic Cells

Human embryonic kidney 293 cells were grown to 70% confluence in 6-wellplates. 2.5 μg of plasmid encoding the t-PA mutant was dissolved in 150μl of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl₂. Added to this (dropwisewhile vortexing) was 150 μl of 50 mM HEPES buffer (pH 7.35), 280 mMNaCl, 1.5 mM NaPO₄, and the precipitate was allowed to form for ten min.at 25° C. The suspended precipitate was then added to the cells in theindividual wells in a 6-well plate and allowed to settle for four hoursin the incubator. The medium was then aspirated off and 1 ml of 20%glycerol in PBS (phosphate buffered saline) was added. The cells werewashed twice, first with 3 ml, then with 1 ml, of serum-free medium.Then 3 ml of fresh medium was added and the cells were incubated forfive days. The medium was then collected and assayed.

When single-chain t-PA was required, the procedure was as describedabove except that plasminogen-depleted serum was used during the growthphase of the cells.

V. Biological Assays A. t-PA Quantitation

The concentration of t-PA in the cell culture supernatants wasdetermined by the ELISA (enzyme linked immunosorbent assay) procedureusing polyclonal antibodies prepared against wild-type t-PA. The amountof t-PA used in each assay described below was based on the results ofthis ELISA procedure.

B. S-2288 Assay

The S-2288 assay was used to measure the proteolytic activity of themutants in the two-chain form. This assay is a direct assay for t-PAproteolytic activity; t-PA cleaves the bond between the small peptideand the paranitroanilide chromophore.

Standard curve samples were prepared by diluting wild-type recombinantt-PA (rt-PA) with cell culture media. The standard curve samples andrt-PA mutant samples were added to the wells of a microtiter plate.Since the assay was used to measure the activity of two-chain rt-PA, anincubation step with human plasmin was included in the procedure. Humanplasmin (KabiVitrum) was added to a final concentration of 0.13 CU(casein units)/ml. The samples were incubated for 90 minutes at roomtemperature.

Aprotinin [Sigma, approximately 14 TIU (trypsin inhibitor unit)/mg] wasadded to a final concentration of 72 μg/ml to inhibit the plasminactivity, and the samples were incubated at room temperature for 15minutes. A 2.16 mM solution of S-2288 was diluted to 1.45 mM with 0.1MTris, 0.106 mM NaCl, 0.02% sodium azide, pH 8.4, and 100 μl of thissolution was added to each well of the microtiter plate (final volume ineach well was 200 μl). Color development was monitored at 405 nm. Theslope of the absorbance vs. time curve for each standard and sample wasdetermined. A standard curve was prepared by plotting the slope of theabsorbance vs. time curve as a function of rt-PA concentration for thert-PA standards. The relative activity concentration of the mutants wasthen determined from the standard curve. The activity concentration ofeach mutant was divided by the concentration for the mutant obtained inthe rt-PA ELISA, and the resulting specific activities were expressedrelative to wild-type t-PA, which was assigned a value of 1.0.

C. S-2251 Assay

This assay is an indirect assay for t-PA activity. In this assay,plasminogen is converted to plasmin by the action of t-PA, and plasmincleaves the S-2251 substrate to release the paranitroanilidechromophore. Production of this chromophore is then measured over time.

1. Fibrin-Stimulated S-2251 Assay

Standard curve samples were prepared as described for the S-2288 assay.Conversion of the samples to the two chain form was accomplished byincubating them with plasmin-Sepharose. Plasmin-Sepharose was preparedby coupling approximately 20.8 CU of human plasmin (KabiVitrum) to 1 mlof cyanogen bromide activated Sepharose (Pharmacia). Theplasmin-Sepharose (50 μl of a 5% slurry) was incubated with shaking for90 min. at room temperature with 150 μl of sample. Following theincubation, the resin was removed by centrifugation, and 10 μl of samplewere added to the wells of a microtiter plate.

Human thrombin (10 μl of a 42 unit/ml solution) was added to each well.The reaction in each well was started by the addition of a cocktail (130μl) composed of 28 μl of human Glu-plasminogen (5.3 μM); 10 μl ofplasminogen-free human fibrinogen (10 μM); 30 μl of 3 mM S-2251(KabiVitrum); and 62 μl of PBS. Color development was monitored at 405nm, and the absorbance at the reference wavelength of 492 nm wassubtracted from each time point. The slope of the absorbance vs. timesquared curve was determined for each standard and mutant sample. Astandard curve was prepared by plotting the slope of the absorbance vs.time squared curve as a function of rt-PA concentration for the rt-PAstandards. The determination of the relative specific activity for themutants was as described for the S-2288 assay.

2. Fibrinogen Stimulated S-2251 Assay

This assay was performed as described for the fibrin-stimulated S-2251assay except that PBS was substituted for the thrombin.

3. Plasma Clot S-2251 Assay

The standard curve sample preparation and the conversion of one-chainrt-PA to two-chain rt-PA using plasmin-Sepharose were as described forthe fibrin-stimulated S-2251 assay. Human thrombin (10 μl of a 31 μg/mlsolution) was added to each well of the microtiter plate. The standardand mutant samples (40 μl) were added to the plate and the reaction wasstarted by adding 100 μl of a mixture of 90 μl of acid citrate dextrosehuman plasma and 10 μl of 9.1 mM S-2251 (KabiVitrum). Color developmentwas monitored at 405 nm and the absorbance at the reference wavelengthof 492 nm was substracted from each time point. The analysis of the datawas as described for the fibrin-stimulated S-2251 assay.

The results of the assays described in sections B and C above arepresented in Table 1, where the asterisks represent the mutants of thisinvention. The remaining mutants have been disclosed previously and areincluded for comparison.

The results of the S-2288 assay indicate that illustrative variants ofthis invention have an activity that is nearly equal to or greater thanthat of wild-type t-PA.

In the fibrin-stimulated S-2251 assay, the single mutants of thisinvention that ar presented in Table 1 have an activity that is similarto that of wild-type t-PA. The multiple mutants E94A,D95A,N117Q andN117Q,D236A,D238A,K240A have substantially higher activity than that ofwild-type t-PA. Surprisingly, their activity was also found to be higherthan that of any of the single mutants.

The results of the fibrinogen-stimulated S-2251 assay show that theactivity of most of the single mutants herein was found to be similar tothat of wild-type t-PA. Unexpectedly, the activity of two of themultiple mutants, E94A,D95A,N117Q and N117Q,D236A,D238A,K240A was foundto be substantially higher than that of the single mutants.

In the plasma-clot S-2251 assay, most of the variants herein were foundto have an activity comparable to that of wild-type t-PA.

                                      TABLE 1                                     __________________________________________________________________________    t-PA              S2251 in                                                                             S2251 with                                                                          S2251 with                                                                          S2288                                    Variant           Plasma Clot                                                                          Fibrin only                                                                         Fibrinogen                                                                          Assay                                    __________________________________________________________________________    Wild-type         1.0    1.0   1.0   1.0                                      *E94A             0.90   0.79  0.74  0.78                                     *D95A             1.07   0.87  0.85  0.98                                     *E94A,D95A        0.91   0.86  0.83  0.91                                     T103N             0.67   0.80  0.47  0.93                                     N117Q             1.10   1.07  1.62  1.01                                     *D236A,D238A,K240A                                                                              1.01   0.87  0.83  0.97                                     *E94A,D95A,T103N  0.63   0.82  0.52  1.09                                     *E94A,D95A,N117Q  1.25   1.51  2.25  1.25                                     *E94A,D95A,D236A,D238A,K240A                                                                    0.67   1.06  0.40  1.95                                     T103N,N117Q       1.12   1.21  1.13  1.19                                     *T103N,D236A,D238A,K240A                                                                        0.64   0.66  0.52  1.12                                     *N117Q,D236A,D238A,K240A                                                                        1.23   1.24  1.55  1.31                                     *D95G             0.99   1.36  1.15  0.95                                     __________________________________________________________________________

D. Plasma Clot Lysis Assay

All t-PA variant samples were converted from the one-chain to thetwo-chain form using plasmin-Sepharose as described for thefibrin-stimulated S-2251 assay above.

The plasma clot lysis assay was performed as follows: 10 ml of 0.15Mcalcium chloride was added to microtiter plate wells. Each well thenreceived 90 μl of centrifuged and 0.45-micron-filtered human citratedplasma pool. The contents were thoroughly mixed to form the plasma clot.Standard samples of rt-PA and the t-PA variants to be assayed werediluted in assay buffer to twice their final concentration (18-800ng/ml). The dilution buffer consisted of 0.1M NaCl, 0.03M sodiumbicarbonate (added fresh just prior to starting the experiment), 4 mMKCl, 1 mM calcium chloride, 1 mM dibasic sodium phosphate, 0.3 mMmagnesium chloride, 0.4 mM magnesium sulfate, 20 mM HEPES(4-[2-hydroxyethyl]-1-piperazineethane sulfonic acid), and 0.01%Polysorbate 80, pH 7.4. Each standard or variant was then mixed with onevolume of the plasma pool. A total of 100 μl of this mixture was thenlayered over the plasma clot after the clot had been allowed to sit atambient temperature for 6-8 hours. The optical density of each plate wasthen read at 405 nm. The plate was then incubated at 37° C. for about 15hours, and the optical density measurement was repeated. For each wellthe difference in optical density values from time 0 to 15 hours wascalculated by subtraction. For the standards, the optical density wasplotted as a function of the log of the concentration of the standard.Unknowns were interpolated from the standard curve. Normalization was toidentically treated wild-type t-PA controls. Standard curves weredetermined using a four parameter fit program. The plate reader employedwas from SLT-Laboratories, Model EAR340AT (Austria).

The results are shown in Table 2, where the asterisks denote thevariants of this invention.

                  TABLE 2                                                         ______________________________________                                        t-PA                Clearance  Clot Lysis                                     Variant             Ratio      in Plasma                                      ______________________________________                                        Wild-type           1.0        1.0                                            *E94A               0.61       0.82                                           *D95A               0.38       0.97                                           *E94A,D95A          0.34       0.74                                           T103N               0.36       0.82                                           N117Q               0.44       0.66                                           *D236A,D238A,K240A  0.45       0.55                                           *E94A,D95A,T103N    0.17       0.57                                           *E94A,D95A,N117Q    0.23       0.70                                           *E94A,D95A,D236A,D238A,K240A                                                                      0.56       0.40                                           T103N,N117Q         0.27       0.86                                           *T103N,D236A,D238A,K240A                                                                          0.26       0.91                                           *N117Q,D236A,D238A,K240A                                                                          0.34       0.45                                           *D95G               0.37       N/A                                            ______________________________________                                         N/A indicates that this data was not available                           

The single variants that are illustrative of this invention haveactivity in this assay comparable to that of wild-type t-PA and T103N,and activity that is better than that of N117Q. Certain of the multiplevariants are comparable in clot lysis activity to wild-type, includingE94A,D95A,N117Q and T103N,D236A,D238A,K240A.

VI. Clearance Rate Assay

Clearance was measured by injecting mice with 125-I-labeled t-PA, andmonitoring the amount of radioactivity remaining in the bloodstream overtime. The preparation of 125-I-labeled t-PA required several steps thatare described below.

A. Radiolabeling of t-PA Variants

The first step in the preparation of the radiolabeled t-PA variants wasto radiolabel the reagent D-Tyr-Pro-Arg-chloromethylketone (YPRck,obtained from Bachem Bioscience, Inc., Philadelphia, Pa.) with 125-I.This reagent acts as a suicide substrate for t-PA by bindingirreversibly to t-PA. The t-PA molecule thus becomes iodinatedindirectly through the covalent bond formed with YPRck.

The YPRck reagent was radiolabeled by Chloramine T catalyzed iodinationusing a method based on that described by Hunter and Greenwood (Nature,194:495 [1962]). In a typical reaction, 50 μl of 1M Tris-HCl at pH 7.5was added to 40 μl of sodium iodide-125 (4 millicuries, 1.8 nmol) in acapped reaction vessel. To this reaction was added 8.3 μl of YPRckreagent (0.83 μg, 1.8 nmol) which had been prepared as a stock solutionof 100 μg/ml in 12 mM HCl. The resulting mixture was a 1:1stoichiometric mixture of sodium iodide and YPRck. The iodinationreaction was initiated by addition of 12.5 μl of 1 mg/ml Chloramine T in0.1M sodium phosphate at pH 7.5. After 60 seconds, the iodinationreaction was terminated by addition of 25 μl of 1 mg/ml sodiummetabisulfite in 0.1M sodium phosphate at pH 7.5. The reaction vesselwas vortexed after each addition. Immediately following the iodinationof YPRck, 2 mls of PBS with 0.01% Tween-20 was added to the reactionvessel and vortexed to dilute the radioactive label.

Cell culture supernatants were collected from 293 cells that had beentransformed six days earlier with DNA encoding the t-PA variants. Thesecells were actively secreting the variant t-PA proteins, and the cellculture medium typically contained about 1 μg/ml of t-PA. Twentymicroliters of the diluted YPRck-125-I reagent was added to 900 μl ofcell culture supernatant. This mixture was incubated at 25° C. for 1hour and then applied to a Sephadex G-25 column previously equilibratedwith 0.1% gelatin in PBS with 0.003% Tween-20. One milliliter fractionswere collected from each column, and the fourth fraction routinelycontained about 85% of the total protein bound radioactivity, asdetermined by trichloroacetic acid precipitation of an aliquot of eachfraction.

B. Pharmacokinetic Assay

The following assay was used to calculate the clearance rate ofYPRck-labeled t-PA variants in mice. Four mice were used to assess eachvariant. The YPRck-labeled-t-PA variants were diluted to a concentrationof 1 million cpm/ml. Each mouse was injected in the tail with 100 μl ofYPRck-labeled-t-PA in a solution of PBS containing 0.5% BSA and 0.01%Tween 20. The mice were tail-bled in pairs. The first pair was bled 1,4, 10, 20, and 30 minutes after the initial injection. The second pairwas bled 2, 7, 15, 25, and 40 minutes after the initial injection. Analiquot of 70 μl of blood was precipitated in 10% trichloroacetic acid(TCA). The TCA precipitable material was counted using a gammascintillation counter and representative results were plotted on agraph, as shown in FIG. 3. The area under the curve (AUC) for each mousewas then calculated. Clearance rate was then determined using theformula: Clearance=Dose/AUC. The clearance rates from the blood of themice that received t-PA variants were normalized to that of wild-typet-PA (clearance rate of wild-type t-PA divided by clearance rate of thevariant).

The results of this assay are presented as clearance ratios and areshown above in Table 2. Illustrative t-PA variants of this inventionhave a clearance ratio that is less than that of wild-type t-PA. Thisindicates that the variants have a decreased clearance as compared towild-type t-PA. Some of the double and triple mutants had a lowerclearance ratio than the single mutants. The mutantsT103N,D236A,D238A,K240A; N117Q,D236A,D238A,K240A; E94A,D95A,T103N; andE94A,D95A,N117Q all displayed lower clearance ratios than the singlemutants at any one of these positions.

We claim:
 1. A DNA molecule encoding a human tissue plasminogenactivator (t-PA) amino acid sequence variant having an alterationconsisting essentially of the substitution of a) a naturally occurringL-alpha-amino acid at at least one of amino acid positions 94 and 95, orb) alanine or glycine at each of amino acid positions 236, 238 and 240,or c) a naturally occurring L-alpha-amino acid at each of amino acidpositions 94 and 95 in combination with either the substitution ofalanine or glycine at each of amino acid positions 236, 238 and 240, orwith the substitution of alanine at each of amino acid positions 296,297, 298 and 299, or d) a naturally occurring L-alpha-amino acid at eachof amino acid positions 94 and 95 or alanine or glycine at each of aminoacid positions 236, 238 and 240 in combination with either thesubstitution of asparagine at amino acid position 103, or with thesubstitution of glutamine at amino acid position 117 of wild-type humant-PA, wherein each of amino acid positions 236, 238 and 240 issubstituted with the same amino acid, and wherein said variant iscapable of converting plasminogen to plasmin and has a decreasedclearance from the bloodstream as compared to wild-type human t-PA. 2.The DNA molecule of claim 1 encoding a human t-PA variant wherein thealteration is the substitution of alanine or glycine for glutamic acidat amino acid position 94 of wild-type human t-PA.
 3. The DNA moleculeof claim 1 encoding a human t-PA variant wherein the alteration is thesubstitution of any naturally occurring L-alpha-amino acid for asparticacid at amino acid position 95 of wild-type human t-PA.
 4. The DNAmolecule of claim 3 encoding a human t-PA variant wherein the alterationis the substitution of alanine or glycine at amino acid position 95 ofwild-type human t-PA.
 5. The DNA molecule of claim 1 encoding a humant-PA variant wherein the alteration is the substitution of alanine foreach aspartic acid at amino acids positions 236 and 238 and for thelysine at amino acid position 240 of wild-type human t-PA.
 6. The DNAmolecule of claim 1 encoding a human t-PA variant wherein the alterationis the substitution of alanine or glycine for glutamic acid at aminoacid position 94 and the substitution of any naturally occurringL-alpha-amino acid for aspartic acid at amino acid position 95 ofwild-type human t-PA.
 7. The DNA molecule of claim 6 encoding a humant-PA variant wherein the alteration is the substitution of alanine orglycine at amino acid position 94 and alanine or glycine at amino acidposition 95 of wild-type human t-PA.
 8. The DNA molecule of claim 7wherein in the encoded human t-PA variant alanine or glycine isadditionally substituted at each of amino acid positions 236, 238 and240 of wild-type human t-PA.
 9. The DNA molecule of claim 7 wherein inthe encoded human t-PA variant asparagine is additionally substituted atposition 103 of wild-type human t-PA.
 10. A DNA molecule encoding ahuman t-PA variant selected from the group consisting of E94A t-PA; D95Xt-PA; E94A,D95A t-PA; D236A,D238A,K240A t-PA; E94A,D95A,T103N t-PA;E94A,D95A,T103N t-PA; E94A,D95A,N117Q t-PA; E94A,D95A,D236A,D238A,K240At-PA; T103N,D236A,D238A,K240A t-PA; and N117Q,D236A,D238A,K240A t-PAwherein X is any naturally occurring L-alpha-amino acid except asparticacid.
 11. A replicable expression vector containing and capable, in atransformed host cell, of expressing the DNA molecule of claim
 1. 12. Areplicable expression vector containing and capable, in a transformedhost cell, of expressing the DNA molecule of claim
 2. 13. A replicableexpression vector containing and capable, in a transformed host cell, ofexpressing the DNA molecule of claim
 3. 14. A replicable expressionvector containing and capable, in a transformed host cell, of expressingthe DNA molecule of claim
 5. 15. A replicable expression vectorcontaining and capable, in a transformed host cell, of expressing theDNA molecule of claim
 6. 16. Host cells transformed with the vector ofclaim
 11. 17. A replicable expression vector containing and capable, ina transformed host cell, of expressing the DNA molecule of claim
 10. 18.The host cells of claim 16 that are eukaryotic cells.
 19. The host cellsof claim 18 that are mammalian.
 20. The host cells of claim 19 that arehuman embryonic kidney 293 cells.
 21. Host cells transformed with thevector of claim
 17. 22. The host cells of claim 21 that are mammalian.23. The host cells of claim 21 that are Chinese hamster ovary (CHO)cells or human embryonic kidney 293 cells.